Protein kinase C (“PKC”) is a key enzyme in signal transduction involved in a variety of cellular functions, including cell growth, regulation of gene expression, and ion channel activity. The PKC family of isozymes includes at least 11 different protein kinases that can be divided into at least three subfamilies based on their homology and sensitivity to activators. Each isozyme includes a number of homologous (“conserved” or “C”) domains interspersed with isozyme-unique (“variable” or “V”) domains. Members of the “classical” or “cPKC” subfamily, α, βI, βII and γPKC, contain four homologous domains (C1, C2, C3 and C4) and require calcium, phosphatidylserine, and diacylglycerol or phorbol esters for activation. In members of the “novel” or “nPKC” subfamily, δ, ε, η and θPKC, a C2-like domain precedes the C1 domain. However, that C2 domain does not bind calcium and therefore the nPKC subfamily does not require calcium for activation. Finally, members of the “atypical” or “αPKC” subfamily, ζ and λ/ιPKC, lack both the C2 and one-half of the C1 homologous domains and are insensitive to diacylglycerol, phorbol esters and calcium.
Studies on the subcellular distribution of PKC isozymes demonstrate that activation of PKC results in its redistribution in the cells (also termed translocation), such that activated PKC isozymes associate with the plasma membrane, cytoskeletal elements, nuclei, and other subcellular compartments (Saito, N. et al., Proc. Natl. Acad. Sci. USA 86:3409-3413 (1989); Papadopoulos, V. and Hall, P. F. J. Cell Biol. 108:553-567 (1989); Mochly-Rosen, D., et al., Molec. Biol. Cell (formerly Cell Reg.) 1:693-706, (1990)). The unique cellular functions of different PKC isozymes are determined by their subcellular location. For example, activated βIPKC is found inside the nucleus, whereas activated βIIPKC is found at the perinucleus and cell periphery of cardiac myocytes (Disatnik, M. H., et al., Exp. Cell Res. 210:287-297 (1994)). εPKC, a member of the novel PKC family independent from calcium but requiring phospholipids for activation, is found in primary afferent neurons both in the dorsal root ganglia as well as in the superficial layers of the dorsal spinal cord.
The localization of different PKC isozymes to different areas of the cell in turn appears due to binding of the activated isozymes to specific anchoring molecules termed Receptors for Activated C-Kinase (“RACKs”). RACKs are thought to function by selectively anchoring activated PKC isozymes to their respective subcellular sites. RACKs bind only fully activated PKC and are not necessarily substrates of the enzyme. Nor is the binding to RACKs mediated via the catalytic domain of the kinase (Mochly-Rosen, D., et al., Proc. Natl. Acad. Sci. USA 88:3997-4000 (1991)). Translocation of PKC reflects binding of the activated enzyme to RACKs and the binding to RACKs is required for PKC to produce its cellular responses (Mochly-Rosen, D., et al., Science 268:247-251 (1995)). Inhibition of PKC binding to RACKs in vivo inhibits PKC translocation and PKC-mediated function (Johnson, J. A., et al., J. Biol. Chem. 271:24962-24966 (1996); Ron, D., et al., Proc. Natl. Acad. Sci. USA 92:492-496 (1995); Smith, B. L. and Mochly-Rosen, D., Biochem. Biophys. Res. Commun. 188:1235-1240 (1992)).
In general, translocation of PKC is required for proper function of PKC isozymes. Peptides that mimic the RACK-binding site on PKC [Ron, D., et al., Proc. Natl. Acad. Sci. USA 92:492-496 (1995); Johnson, J. A., et al., J. Biol. Chem. 271:24962-24966 (1996)] are isozyme-specific translocation inhibitors of PKC that selectively inhibit the function of the enzyme in vivo. Such isozyme-selective inhibitors of PKC have been identified based on their ability to selectively inhibit the interaction of the activated isozymes with their respective anchoring proteins (RACKs) (Souroujon, M. and Mochly-Rosen, D., Nature Biotechnol. 16:919-924, (1998)). These short peptide inhibitors (7-12 amino acids long) have been shown to selectively interfere with the functions of individual isozymes (Mochly-Rosen, D., et al., Proc. Natl. Acad. Sci. USA 88:3997-4000, (1991); Ron, D., et al., J. Biol. Chem. 270:24180-24187, (1995); Johnson, J. A., et at, J. Biol. Chem. 271:24962-24966, (1996); Zhang, Z., et al., Biophys. J. 70(2, part 2):A391, (1996); Gray, M. O., et al., J. Biol. Chem. 272:30945-30951, (1997)).
Translocation agonist peptides of β and εPKC, as well as other PKC isozymes, have also been identified [Ron, D. and Mochly-Rosen, D., Proc. Natl. Acad. Sci. USA 92:492-496, (1995); Dorn, G. W., et al., Proc. Natl. Acad. Sci. USA 96:12798-12803, (1999)]. Peptides that mimic the PKC-binding site on RACKs [Mochly-Rosen, D., et al., J. Biol. Chem. 226:1466-1468 (1991); Mochly-Rosen, D., et al., Science 268:247-251 (1995)] are isozyme-specific translocation activators of PKC that selectively inhibit the function of the enzyme in vivo (Mochly-Rosen, D., Proc. Natl. Acad. Sci. USA 92:492-496, (1995); Dorn, G. W., et al., Proc. Natl. Acad. Sci. USA 96:12798-12803, (1999)). These 6-8 amino acid peptides derived from PKC are homologous to a sequence within their corresponding RACK and hence they were termed pseudo-βRACK (ΨβRACK) and pseudo-εRACK (ΨεRACK), respectively. Introduction of ΨβRACK or ΨεRACK into cells causes a selective translocation of the corresponding isozymes and increases their catalytic activity as measured by substrate phosphorylation in vitro and in vivo (Ron, D. and Mochly-Rosen, D., Proc. Natl. Acad. Sci. USA 92:492-496, (1995); Dorn, G. W., et al., Proc. Natl. Acad. Sci. USA 96:12798-12803, (1999)). The position in the C2 or C2-like domain of the ΨRACK sequence in isozymes whose RACK has not been identified yet (e.g., δ and θPKC) was also found to correspond to translocation agonist peptides [Chen et al., Proc. Natl. Acad. Sci. USA 98:1114-1119, (2001)]. For example, introduction of ΨδRACK into cells causes a selective translocation of the δPKC and increases its catalytic activity [Chen et al., Proc. Natl. Acad. Sci. USA 98:1114-1119, (2001)]. These peptides have also been used to identify the role of βPKC, δPKC and εPKC in cells and in vivo [Ron, D. and Mochly-Rosen, D., Proc. Natl. Acad. Sci. USA 92:492-496, (1995); Chen et al., Proc. Natl. Acad. Sci. USA 98:1114-1119, (2001); Dorn, G. W., et al., Proc. Natl. Acad. Sci. USA 96:12798-12803, (1999)).
From a therapeutic perspective, individual isozymes of PKC have been implicated in the mechanisms of various disease states, including the following: cancer (alpha and delta PKC); cardiac hypertrophy and heart failure (beta I and beta II PKC) nociception (gamma and epsilon PKC); ischemia including myocardial infarction (epsilon and delta PKC); immune response, particularly T-cell mediated (theta PKC); and fibroblast growth and memory (delta and zeta PKC). Various PKC isozyme- and variable region-specific peptides have been previously described (see, for example, U.S. Pat. No. 5,783,405). The role of εPKC in pain perception has recently been reported (WO 00/01415; U.S. Pat. No. 6,376,467) including therapeutic use of the εV1-2 peptide, a selective inhibitor of εPKC first described in the U.S. Pat. No. 5,783,405.
It is clear that PKC isozymes are involved in a variety of disease states, and there continues to be a need for methods of modulating the action of specific PKC isozymes to develop therapeutic agents to treat human disease.